Large scale processes have been developed and are being used commercially to convert glycerol esters of fatty acids (also known as glycerides, mono-glycerides, di-glycerides, and tri-glycerides) to glycerol esters of methanol, ethanol, or other alcohols. The resulting fatty acid esters (also known as FAME for fatty acid methyl ester or FAEE for fatty acid ethyl ester) are commonly known as “biodiesel”, because they can be used by themselves or in blends with conventional hydrocarbons as fuel for diesel engines. The raw materials for the synthesis of biodiesel can include vegetable oil, animal fats, and discarded cooking oil. A major volume byproduct of the biodiesel process is glycerol (also known as glycerin or glycerine). For each kilogram of biodiesel produced, about 0.1 kilogram of glycerol byproduct is produced.
When the catalyst for biodiesel synthesis is sodium hydroxide or potassium hydroxide, the glycerol byproduct is typically about 80% to 90% glycerol by weight, with the remainder of the byproduct being mostly water, methanol or ethanol (depending on which alcohol was used for the transesterification), various salts, and low levels of other organic compounds. The raw glycerol byproduct is alkaline and viscous, so it is usually neutralized down to a pH of about 4 or 5 with sulfuric acid, hydrochloric acid, or other acid, which reduces the viscosity and leaves the presence of the resulting salts, such as sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, etc., with the exact composition obviously depending on the compounds used in the process. Much or all of the alcohol can typically be removed and recovered from the crude glycerol by distillation. The sodium or potassium hydroxide catalyst used in this type of process is called a homogeneous catalyst.
Another process for producing biodiesel relies on a “heterogeneous catalyst”. An example of this is called Esterfip-H, commercialized by the French company Axens. The exact nature of this catalyst is proprietary, but it is reported to be a spinel mixed oxide of two non-noble metals, and it is reported to give a much cleaner glycerol byproduct than that from a homogeneous catalyst. The glycerol byproduct from a heterogeneous catalyst is reported to be 98% pure and free of salts (Ondrey, 2004).
With the growth of the biodiesel business there has of necessity been a parallel growth in the volume of glycerol byproduct. Some of the crude glycerol byproduct from biodiesel industry is purified by distillation and used in various industries that have classically used glycerol as a feedstock, and the rest of the glycerol from the biodiesel industry is considered to be a burdensome waste product. As a result, the value of the crude glycerol has plummeted to $0.05/lb or less in recent years (De Guzman, 2010). As such, glycerol has become a potentially inexpensive alternative to sugars and other carbohydrates such as glucose, fructose, sucrose, maltose, starch, and inulin, as a fermentation feedstock for production of fuels and chemicals (Clomburg and Gonzalez, 2010; Yazdani and Gonzalez, 2007). As of this writing, glucose and sucrose cost about $0.15 to $0.25/lb, and therefore a fermentation process that can use glycerol instead of other sugars could result in a substantial economic advantage.
A number of microorganisms have been developed for the commercial production of useful chemicals via fermentation using renewable sugars. Escherichia coli (E. coli) strains capable of producing organic acids in significant quantities using a variety of sugars as the source of carbon are well known in the art. For example, the U.S. Patent Application Publication No. 2009/0148914 provides strains of E. coli as a biocatalyst for the production of chemically pure acetate and/or pyruvate. The U.S. Pat. No. 7,629,162 provides derivatives of E. coli KO11 strain constructed for the production of lactic acid. International Patent Application Nos. WO 2008/115958 and WO 2010/115067 published under the Patent Cooperation Treaty provide microorganism engineered to produce succinate and malate in a minimal salt medium containing glucose as a source carbon in pH-controlled batch fermentation. U.S. Pat. No. 7,241,594 and U.S. Pat. No. 7,470,530 and the International Patent Application Publication No. WO 2009/024294 provides rumen bacterium Mannheimia succiniproducens useful in the fermentative production of succinic acid using sugars as the source of carbon. U.S. Pat. Nos. 5,000,000, 5,028,539, and 5,424,202 provide E. coli strains for the production of ethanol. U.S. Pat. Nos. 5,482,846, 5,916,787, and 6,849,434 provide gram-positive microbes for ethanol production. U.S. Pat. No. 7,098,009 provides Bacillus strains for the production of L(+) lactic acid. U.S. Pat. Nos. 7,223,567, 7,244,610, 7,262,046, and 7,790,416 provide E. coli strains for the production of succinic acid.
Most of the microbial organisms currently used in the biotechnology industry for the production of fuels and chemicals have a dedicated metabolic pathway for glycerol utilization. However, none of these industrial microorganisms have ever been shown to have the capacity to use glycerol as a feedstock with production parameters that are attractive for commercial manufacturing. This inability of the industrial microorganism to utilize glycerol as a commercial feedstock in the manufacturing of useful chemicals is attributed to certain regulatory metabolic feedback control mechanisms that are operational within the microbial cells.
The uptake and metabolism of glycerol by microorganisms has been well studied, particularly in Escherichia coli (Lin, 1996; Gonzalez et al., 2008). As shown in FIG. 1, in E. coli, glycerol enters the cell by a facilitated diffusion protein encoded by the glpF gene. In the “classical” glycerol metabolic pathway, glycerol is phosphorylated by glycerol kinase, encoded by the glpK gene, to give glycerol-3-phosphate (G3P). The G3P is then reduced to dihydroxyacetone phosphate by either the G3P dehydrogenase encoded by glpD or the three-subunit G3P dehydrogenase encoded by glpABC. The GlpK-GlpD/GlpABC pathway is considered to be respiratory route as it requires electron acceptors and is believed to be operational under aerobic conditions or when an alternative electron acceptor is present, such as nitrate or fumarate.
Another pathway for glycerol metabolism within the microbial cell is referred to as the non-classical pathway and is thought to be operational under anaerobic conditions (Gonzalez et al., 2008). In this second pathway, glycerol transported into the cell is reduced to dihydroxyacetone by a glycerol dehydrogenase encoded by gldA. The dihydroxyacetone is then phosphorylated by a phosphoenolpyruvate-dependent dihydroxyacetone kinase encoded by dhaKLM. The dihydroxyacetone phosphate resulting from either of these pathways can enter into the glycolytic pathway through triose phosphate isomerase, encoded by tpi. Triosephosphate isomerase converts dihydroxyacetone phosphate into glyceraldehyde-3-phosphate which can enter into the tricarboxylic acid pathway after conversion into glycerate-1,3-diphosphate which in turn is converted into phosphoenolpyruvate.
There are several reports that disclose microbial production of various compounds by fermentation from glycerol. In general, the chemicals produced via microbial fermentation from glycerol, including succinate, ethanol, 1,2-propanediol, hydrogen, and formate, are produced at titers that do not appear to be high enough to compete with other known commercial processes for producing those compounds (Gonzalez et al., 2008; Durnin et al., 2009; Yazdani and Gonzalez, 2008).
Blankenschein et al. (2010) described an engineered E. coli strain that is contains ΔadhE, Δpta, ΔpoxB, ΔldhA, Δppc, and a plasmid pZS-pyc that over-expresses pyruvate carboxylase from Lactococcus lactis. Preliminary experiments with GldA-DhaKLM expressed from a separate vector in the ΔadhE, Δpta, ΔpoxB, ΔldhA, Δppc, [pZS-pyc] stain showed no improvements in succinate production. There are certain disadvantages with this glycerol utilizing strain of E. coli. This strain, which was not given a specific name, produced only 14 g/l of succinate in 72 hours with a yield of 0.69 g/g glycerol. Moreover, the plasmid pZS-pyc requires chloramphenicol for maintenance and anhydrotetracycline for induction, both of which are undesirable for large scale fermentations.
Yazdani and Gonzalez (2008) describe two E. coli strains, SY03 and SY04, designed to produce ethanol plus hydrogen or formate, respectively. These two strains also require the plasmid pZSKLMGldA. This plasmid is designed to over express the E. coli dhaKLM operon and gldA, which presumably increases flux through the “non-classical” glycerol pathway. In the most favorable example given, SY04 containing pZSKLMGldA produced about 10 g/l ethanol and 9 g/l formate from about 22 g/l glycerol, in 100 hours. These fermentation parameters are not high enough for a competitive commercial process. Moreover, the pZSKLMGldA plasmid requires chloramphenicol for maintenance and anhydrotetracycline for induction, both of which are undesirable for large scale fermentations.
The International Patent Application Publication No. WO 2010/051324 discloses E. coli strains with the plasmids LA01 (pZSglpKglpD) and LA20 (pZSglpKglpD) overexpressing glpK and glpD genes to produce D-lactate and L-lactate, respectively.
Zhang et al. (2010) have described an engineered E. coli strain, XZ721, that contains a mutation in the promoter region of pck gene (called pck*), ΔptsI, and ΔpflB. In fermentors, strain XZ721 using glycerol as a source of carbon produced 12 g/l succinate in 6 days, with a yield of 0.80 mol/mol glycerol used, which is equivalent to 1.02 g/g glycerol used. Deletion of gldA or dhaM in the pck* background led to higher succinate titers (13.2 g/l and 12.7 g/l respectively), suggesting that the GldA-DhaKLM route might not be the preferred pathway for succinate production under the fermentation conditions used.
Scholten et al. have recently isolated a novel ruminant bacterium in the Pasteurellaceae family that was named DD 1 or Basfia succiniciproducens. The DD 1 bacterium produces succinate from glycerol anaerobically (US Patent Application 2011/0008851). However, Basfia succiniciproducens does not grow on a minimal medium without added nutrients, and the maximum reported titer was 35 g/l succinate from glycerol as the sole carbon source. If maltose was added to the medium, the titer was improved to 58 g/l, but a significant amount of glycerol remained unused.
Trinh and Srienc (2009) reported improving the production of ethanol from glycerol by using elementary mode analysis to design an optimal E. coli strain. The optimal strain, TCS099 was then constructed, with a genotype of Δzwf, Δndh, ΔsfcA, ΔmaeB, ΔldhA, ΔfrdA, ΔpoxB, Δpta, and Δmdh. After metabolic evolution, TCS099 containing a plasmid, pLOI297, which expressed Zymomonas mobilis ethanol production genes, was able to produce ethanol from glycerol at up to 97% of theoretical yield and titer of about 17 g/l from 40 g/l glycerol. However, this process would again not be economically competitive with other current processes. The authors pointed out that mutations in glycerol kinase can increase the specific growth rate of strains on glycerol, as was known in 1970 (Zwaig et al., 1970), and they suggested that their evolved strain might have generated an increase in flux through glycerol kinase through a mutation, but they did not sequence or characterize the glycerol kinase gene in their evolved strain, and they did not suggest that deliberate introduction of a mutated glycerol kinase would increase rate of ethanol production or lead to a higher ethanol titer with glycerol as the source of carbon.
Since the industrial scale microbial production of biofuels and organic chemicals is carried out under anaerobic fermentative conditions, it is logical to activate the anaerobic glycerol utilization pathway inside the microbial cell in order to make the microorganisms to utilize glycerol as the feedstock. But as described above, the genetic manipulation of the anaerobic glycerol utilization pathway has not produced expected improvements in the production of desired chemicals using glycerol as the sole source of carbon. There has been disclosures in the prior art of feedback resistant alleles of glpK and of regulation of expression of glycerol utilization genes in the aerobic glycerol utilization pathway by the repressor protein coded by glpR gene. However, no effort has ever been made to improve the production of commercially useful chemicals from glycerol by replacing the wild type glpK allele in a production strain with a feedback resistant glpK allele or by deleting the repressor of glycerol utilization, such as the E. coli glpR gene, or a combination of the two approaches. The present inventors have surprisingly found out that by means of engineering the GlpK-GlpD/GlpABC route for glycerol utilization followed by a process of metabolic evolution in the microbial cells selected for the production of succinic acid, it is possible to confer the ability to utilize glycerol as the source of carbon, while retaining the original production capacity for succinic acid. Although the present invention is explained in detail with the construction of an E. coli strain suitable for the commercial production of succinic acid using glycerol as the source of carbon, the general theme and the spirit of the present invention can be applied in the construction of the microbial strains for the production of a number of other commercially useful chemicals using glycerol as the source of carbon in a microbial fermentation process.